Silvaticusins A–D: ent-kaurane diterpenoids and a cyclobutane-containing ent-kaurane dimer from Isodon silvaticus

Three new ent-kaurane diterpenoids, silvaticusins A–C (1–3), along with a new ent-kaurane dimer silvaticusin D (4) were isolated from the aerial parts of Isodon silvaticus. The structures of these new compounds were established mainly by comprehensive analysis of their NMR and MS data. The absolute configuration of compounds 1 and 4 were determined using a single-crystal X-ray diffraction and computational methods, respectively. Compounds 2 and 3 were found to exhibit remarkable cytotoxic effects against five human tumor cell lines (HL-60, A-549, SMMC-7721, MDA-MB-231, and SW-480), with IC50 values spanning from 1.27 ± 0.08 to 7.52 ± 0.33 μM. Graphical Abstract Supplementary Information The online version contains supplementary material available at 10.1007/s13659-024-00465-9.


Introduction
Isodon species, highly esteemed for their medicinal virtues, harbor a rich array of diterpenoids, with ent-kauranoids being the most prominent class [1,2].The recent discovery of labdane-based meroditerpenoids with cyclobutane moieties, including isoscopariusins B and C [3], along with clerodane-based dimers scospirosins A and B [4], and scoparicacids A-C [5] from I. scoparius, further highlights the chemical versatility of these plants.Furthermore, the bioactivities of these compounds are particularly notable.For example, eriocalyxin B, derived from I. eriocalyx var.laxiflora, has recently been found to demonstrate significant therapeutic potential in oncology by effectively inhibiting cell migration in triple-negative breast cancer [6].
In a concerted effort to uncover natural products with distinctive structures and potent bioactivities from the genus Isodon, a comprehensive phytochemical study was conducted on Isodon silvaticus collected from Changdu in the Tibet Autonomous Region.To date, there has been a scarcity of information regarding the chemical composition of I. silvaticus, except that a reported synthetic derivative acetyl-macrocalin B [7] has been isolated from this plant.This compound has undergone an in-depth pharmacological assessment, revealing its remarkable capacity to inhibit the growth of non-small cell lung cancer and esophageal squamous cell carcinoma in both in vitro and in vivo models [8,9].The current study resulted in the isolation of silvaticusins A-D (1-4) (Fig. 1), involving two new C-20-oxygenated-ent-kaurane diterpenoids (1 and 2), a new C-20 non-oxygenated-ent-kaurane diterpenoid (3), and a new dimeric diterpenoid (4) characterized by a cyclobutane moiety formed through a [2 + 2] cycloaddition between two 6,7-seco-ent-kaurane diterpenoids.Meanwhile, 37 miscellaneous known diterpenoids were characterized from the same plant material (Fig. S44 and Table S13).This study presents the first characterization of new compounds from I. silvaticus.Notably, the discovery of silvaticusin D (4) enriches the limited array of ent-kaurane dimers with a cyclobutane ring.This group now includes maoecrystal M (7,20-epoxyent-kaurane dimer) [10], bisjaponins A and B (6,7-secoent-kaurane dimer, 6,7-seco-/7,20-epoxy-ent-kaurane dimer, respectively) [11], and bistenuifolin L and M (C-20 non-oxygenated ent-kaurane dimers) [12].The cyclobutane moiety in these compounds, formed through [2 + 2] cycloaddition, is a key structural feature that may confer unique biological properties and synthetic challenges, underscoring their importance in the field of natural product chemistry [13,14].The structures of silvaticusins A-D (1-4) were determined through a combinatorial use of spectroscopic analysis, X-ray diffraction, and quantum chemical calculations.Notably, compounds 2 and 3 were found to exhibit remarkable cytotoxic activities against HL-60, A-549, SMMC-7721, MDA-MB-231, and SW-480 human tumor cell lines with IC 50 ranging from 1.27 ± 0.08 to 7.52 ± 0.33 μM.Herein, the structure elucidation and bioactivity evaluation of silvaticusins A-D (1-4) were reported.
Previous research on diterpenoids from the Isodon genus has shown that the exo-methylene cyclopentanone D-ring system is crucial for their antitumor and anti-inflammatory effects, with C-14 hydroxylation usually enhancing these effects [21].In this research, compounds 2 and 3, which possess this D-ring system, were evaluated for cytotoxicity against HL-60, A-549, SMMC-7721, MDA-MB-231, and SW-480 cell lines using the MTS assay [22], with cisplatin and paclitaxel as positive controls.Compounds 2 and 3 exhibited significant cytotoxic activity, with IC 50 values ranging from 1.27 ± 0.08 to 7.52 ± 0.33 μM for all five cell lines (Table 4).

General experimental procedures
General experimental procedures can be found in the Supplementary Material (Part 1).

Plant material
The source of the plant material can be found in the Supplementary Material (Part 2).

Extraction and isolation
The detailed procedures for extraction and isolation can be found in in the Supplementary Material (Part 3).

X-ray crystal structure analysis
The X-ray crystal structure analysis of silvaticusin A (1) can be found in in the Supplementary Material (Part 4).

The cytotoxicity assay
The cytotoxicity assay has been described previously [23].

Table 1 1
H NMR spectroscopic data of silvaticusins A-C (1-3) (δ in ppm, J in Hz) Recorded on an 800 MHz NMR spectrometer in pyridine-d 5 a Recorded on a 500 MHz NMR spectrometer in pyridine-d 5 .b

Table 2
13C NMR spectroscopic data of silvaticusins A-C (1-3) (δ in ppm) a Recorded on a 500 MHz NMR spectrometer in pyridine-d 5 .b Recorded on a 600 MHz NMR spectrometer in pyridine-d 5

Table 4
Cytotoxic activities of compounds 2 and 3 against five human tumor cell linesResults are expressed as IC 50 values in μM.Cisplatin and paclitaxel were used as positive controls